The Inhibition of Protein Kinase C ß Contributes to the Pathogenesis of Preeclampsia by Activating Autophagy.

BACKGROUND: Preeclampsia is a devastating hypertensive disorder of pregnancy with unknown mechanism. Recent studies have considered abnormal autophagy as a new cellular mechanism for this disorder, while little is known about how autophagy is specifically involved and what factors are implicated. Here, we report a previously unrecognized preeclampsia-associated autophagic regulator, PKCß, that is involved in placental angiogenesis. METHODS: PKCß levels were evaluated by quantitative real-time PCR, western blotting, immunofluorescence and by the analysis of public data. The autophagy-regulating role of PKCß inhibition in preeclampsia pathogenesis was studied in a mouse model, and in human umbilical vein endothelial cells (HUVECs) and human choriocarcinoma cells (JEG-3). FINDINGS: PKCß was significantly downregulated in human preeclamptic placentas. In a mouse model, the selective inhibition of PKCß by Ruboxistaurin was sufficient to induce preeclampsia-like symptoms, accompanied by excessive autophagic flux and a disruption in the balance of pro- and anti-angiogenic factors in mouse placentas. In contrast, autophagic inhibition by 3-methyladenine partially normalized hypertension, proteinuria and placental angiogenic imbalance in PKCß-inhibited mice. Our in vitro experiments demonstrated that PKCß inhibition activated autophagy, thus blocking VEGFA-induced HUVEC tube formation and resulting in the significant upregulation of sFLT1 and downregulation of VEGFA in JEG-3 cells. INTERPRETATION: These data support a novel model in which autophagic activation due to PKCß inhibition leads to the impairment of angiogenesis and eventually results in preeclampsia. FUNDING: Shanghai Key Program of Clinical Science and Technology Innovation, National Natural Science Foundation of China and Shanghai Medical Center of Key Programs for Female Reproductive Diseases.

Glycogen Synthase Kinase 3 Inactivation Drives T-bet-Mediated Downregulation of Co-receptor PD-1 to Enhance CD8(+) Cytolytic T Cell Responses.

Despite the importance of the co-receptor PD-1 in T cell immunity, the upstream signaling pathway that regulates PD-1 expression has not been defined. Glycogen synthase kinase 3 (GSK-3, isoforms α and ß) is a serine-threonine kinase implicated in cellular processes. Here, we identified GSK-3 as a key upstream kinase that regulated PD-1 expression in CD8(+) T cells. GSK-3 siRNA downregulation, or inhibition by small molecules, blocked PD-1 expression, resulting in increased CD8(+) cytotoxic T lymphocyte (CTL) function. Mechanistically, GSK-3 inactivation increased Tbx21 transcription, promoting enhanced T-bet expression and subsequent suppression of Pdcd1 (encodes PD-1) transcription in CD8(+) CTLs. Injection of GSK-3 inhibitors in mice increased in vivo CD8(+) OT-I CTL function and the clearance of murine gamma-herpesvirus 68 and lymphocytic choriomeningitis clone 13 and reversed T cell exhaustion. Our findings identify GSK-3 as a regulator of PD-1 expression and demonstrate the applicability of GSK-3 inhibitors in the modulation of PD-1 in immunotherapy.

Combination of long-acting HIV fusion inhibitor albuvirtide and LPV/r showed potent efficacy in HIV-1 patients.

BACKGROUND: Long acting antiretroviral drugs represent a promising approach for chronic treatment of HIV infection. Here, we study the efficacy and safety of albuvirtide (ABT), an HIV-1 fusion inhibitor with a half life of 11-12 days in human. METHODS: ABT was evaluated in a 7-week, open-label and randomized trial, combining with LPV/r. Twenty HIV-1-infected adults were assigned to two dose groups, receiving ABT (160 or 320 mg) given weekly and LPV/r given twice daily. RESULTS: At week 7, the decline of HIV-1 RNA from baseline was 1.9 (1.3-2.3) log10 and 2.2 (1.6-2.7) log10 copies/ml, and suppression of HIV-1 RNA to below 50 copies/ml was achieved in 11.1 % (1/9) and 55.6 % (5/9) patients, for the 160 and 320 mg dose group respectively. CONCLUSION: A clear dose-efficacy correlation of ABT was demonstrated. ABT combining with LPV/r is a promising two-drug regimen to be tested in larger patient population.

Oral protein kinase c ß inhibition using ruboxistaurin: efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with diabetic retinopathy in the Protein Kinase C ß Inhibitor-Diabetic Retinopathy Study and the Protein Kinase C ß Inhibitor-Diabetic Retinopathy Study 2.

PURPOSE: To evaluate efficacy, safety, and causes of vision loss among 813 patients (1,392 eyes) with moderately severe to very severe nonproliferative diabetic retinopathy from the Protein Kinase C ß Inhibitor-Diabetic Retinopathy Study and Protein Kinase C ß Inhibitor-Diabetic Retinopathy Study 2 ruboxistaurin (RBX) protein kinase C ß inhibitor trials. METHODS: Patients in these 3-year, randomized, placebo-controlled, double-masked, Phase 3 trials had best-corrected Early Treatment Diabetic Retinopathy Study visual acuity ≥45 letters (∼20/125 Snellen), Early Treatment Diabetic Retinopathy Study retinopathy level 47A/B-53E, and no previous panretinal photocoagulation in ≥1 eye. Patients received placebo (N = 401) or RBX 32 mg/day (N = 412). Data from the 2 studies were combined and masked evaluation of retinal photographs was performed for cause of visual decline in all patients experiencing sustained moderate visual loss (≥15-letter loss sustained for the last 6 months of study). RESULTS: In the studies combined, sustained moderate visual loss occurred in 10.2% of placebo-treated patients versus 6.1% of RBX-treated patients (P = 0.011). A ≥15-letter gain occurred in 2.4% of placebo versus 4.7% of RBX eyes (P = 0.021) and a ≥15-letter loss occurred in 11.4% versus 7.4%, respectively (P = 0.012). Diabetic macular edema was the probable primary cause of vision loss. Among eyes without focal/grid photocoagulation at baseline, fewer RBX group eyes (26.7%) required initial focal/grid photocoagulation versus placebo (35.6%; P = 0.008). No safety concerns were identified. CONCLUSION: Analysis of data combined from two similar studies adds further statistical significance to RBX's beneficial effects on visual loss, need for focal laser, and vision gain, most likely through effects on macular edema.

[State of the liver antioxidant system and content of matrix metalloproteinase-2 of the large intestine under the effect of maleimide derivative in experimental colorectal carcinogenesis in rats].

The maleimide derivative--1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity did not cause substantial changes of liver antioxidant system and level of matrix metalloproteinase-2 in intestinal mucosa after chronic treatment (for 20 weeks). MI-1 did not cause significant changes in the content of thiobarbituric-active products and plasma membrane protein carbonyl groups in the rat liver. However activities of superoxide dismutase, glutathione peroxidase, and content of reduced glutathione were decreased in both doses--0.027 and 2.7 mg/kg. The level of matrix metalloproteinase-2 in intestinal mucosa was decreased just in maximum dose--2.7 mg/kg. The contents of thiobarbituric-active products, protein carbonyl groups, reduced glutathione, matrix metalloproteinase-2, activities of glutathione peroxidase and glutathione-S-transferase in the liver cells have increased in 1.2-dimethylhydrazine-induced colon cancer in rats. The activities of enzymes of the first line of antioxidant defense--superoxide dismutase and catalase were decreased to 40%. The maleimide derivative prevents development of oxidation stress and partially reduce them to control level.

[Effect of maleimide derivative on the morpho-functional state of the kidney in experimental colon carcinogenesis in rats].

The effect of long-term administration of the novel maleimide derivative with antiproliferative activity on kidney morpho-functional state in experimental (1,2-dimethylhydrazine-induced) colon carcinogenesis has been investigated on 60 male rats. 1,2-Dimethylhydrazine was injected subcutaneously in dose 20 mg/kg one time per week during 20 weeks. Maleimide derivative in dose of 0,027 and 2,7 mg/kg was given per os daily during the same time. The state of kidneys was evaluated after morphometrical investigation and measurement of urea, creatinine and chlorides levels in blood serum. It hasn't been revealed significant structure-functional changes in kidneys after daily administration during 20 weeks. The induction of proliferate activity of distal tubules epithelial cells and reducing of epithelial layer thickness in proximal tubules in kidneys in rats with experimental carcinogenesis has been observed. In experimental colon carcinogenesis, maleimide derivative displays some protective action to tubular apparatus of rat's kidney cortical nephrons and reduces the frequency of preneoplastic changes in tubules epithelial cells.

[Evaluation of hepatotoxicity of novel maleimide derivative with cytostatic activity and its effect on peroxidation process and antioxidant system in the liver].

Novel maleimide derivative 1-(4-Cl-benzyl)-3-Cl-4-(CF3-phenylamino)-1H-pyrrol-2.5-dione (MI-1) with cytostatic activity does not damage rat liver cells after intragastric administration. It is confirmed by alanine- and aspartate aminotransferase, lactate dehydrogenase and alkaline phosphatase activities in blood serum. Preliminary treatment with MI-1 partially prevents from liver cell damage caused by CoCl2. the content of thiobarbituric-active products, protein carbonyl groups, reduced glutathione and activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in the liver cell after one- and ten-days treatment with novel maleimide derivative have been studied. It has been determined that one-day administration of MI-1 has not caused significant changes of peroxidation process and antioxidant system in the liver cells. After ten days treatment the activity of glutathione-S-transferase has been increased, superoxide dismutase--two times decreased, but other parameters have not been significantly changed. Ten days injection of CoCl2 provokes some manifestations of oxidative stress in the liver cells that has been partially leveled by preliminary treatment with maleimide derivative.

Pharmacokinetics and tolerability of a novel long-acting glucagon-like peptide-1 analog, CJC-1131, in healthy and diabetic subjects.

OBJECTIVE: The safety, tolerability, pharmacokinetics and preliminary pharmacodynamics of single rising doses of a novel GLP-1 analog, CJC-1131, was evaluated. METHODS: CJC-1131 was subcutaneously injected in 8 groups (1.5 - 20.5 microg/kg) of healthy subjects (each group of six subjects included 1 placebo per dose level). CJC-1131 was also injected subcutaneously in 6 groups (1.5 - 12 microg/kg) of Type 2 diabetic patients after a 9-day washout period from their own anti-diabetic medication. Each group of 8 patients included 2 placebo-treated patients. Seven blood glucose measurements were taken daily, and meal tolerance tests were performed on the day before dosing and on Day 3. RESULTS: CJC-1131 was quickly absorbed from the subcutaneous space, and a less than dose-proportional increase was found in Cmax. The half-life of CJC-1131 varied from 8.9 - 14.7 days in healthy subjects and from 9.1 - 13.8 days in patients. The maximum tolerated dose in healthy subjects was established at 12 microg/kg with nausea and vomiting being the dose-limiting events. These events occurred generally in the morning after dosing. Blood glucose levels in patients decreased on Day 1 in proportion with dose, with a maximum average decrease of 4.1 mmol/l in the highest dose group. Higher doses appeared to be related to a slight weight loss in patients. CONCLUSIONS: Conjugation to albumin led to a major prolongation of the half-life of GLP-1. The tolerability of this potential antidiabetic drug seems to be limited only by gastrointestinal complaints.

Effect of ruboxistaurin in patients with diabetic macular edema: thirty-month results of the randomized PKC-DMES clinical trial.

OBJECTIVE: To evaluate the safety and efficacy of orally administered ruboxistaurin (RBX) as a mesylate salt in patients with diabetic macular edema (DME). DESIGN: Multicenter, double-masked, randomized, placebo-controlled study of 686 patients receiving placebo or RBX orally (4, 16, or 32 mg/d) for 30 months. At baseline, patients had DME farther than 300 mum from the center of the macula, an Early Treatment Diabetic Retinopathy Study retinopathy severity level from 20 to 47A without prior photocoagulation, and an Early Treatment Diabetic Retinopathy Study visual acuity of 75 or more letters in the study eye. The primary study outcome was progression to sight-threatening DME or application of focal/grid photocoagulation for DME. Main Outcome Measure Masked grading of stereoscopic fundus photographs. RESULTS: The delay in progression to the primary outcome was not statistically significant (32 mg of RBX vs placebo, P = .14 [unadjusted]; Cox proportional hazards model adjusted for covariates, hazards ratio = 0.73; 95% confidence interval, 0.53-1.0; P = .06). However, application of focal/grid photocoagulation prior to progression to sight-threatening DME varied by site, and a secondary analysis of progression to sight-threatening DME alone showed that 32 mg of RBX per day reduced progression, compared with placebo (P = .054 [unadjusted]; Cox proportional hazards model, hazards ratio = 0.66; 95% confidence interval, 0.47-0.93; P = .02). CONCLUSIONS: Although progression to the primary outcome was not delayed, daily oral administration of RBX may delay progression of DME to a sight-threatening stage. Ruboxistaurin was well tolerated in this study.

A multicenter clinical study of the safety and activity of maleimide-polyethylene glycol-modified Hemoglobin (Hemospan) in patients undergoing major orthopedic surgery.

BACKGROUND: Hemospan (Sangart Inc., San Diego, CA), a polyethylene glycol-modified hemoglobin with unique oxygen transport properties, has successfully completed a phase I trial in healthy volunteers. Because adverse events are expected to increase with age, the authors conducted a phase II safety study of Hemospan in elderly patients undergoing elective hip arthroplasty during spinal anesthesia. METHODS: Ninety male and female patients, American Society of Anesthesiologists physical status I-III, aged 50-89 yr, in six Swedish academic hospitals were randomly assigned to receive either 250 or 500 ml Hemospan or Ringer's acetate (30 patients/group) before induction of spinal anesthesia. Safety assessment included vital signs and Holter monitoring from infusion to 24 h, evaluation of laboratory values, and fluid balance. The hypothesis to be tested was that the incidence of adverse events would be no more frequent in patients who received Hemospan compared with standard of care (Ringer's acetate). RESULTS: Three serious adverse events were noted, none of which was deemed related to study treatment. Liver enzymes, amylase, and lipase increased transiently in patients in all three groups. There were no significant differences in electrocardiogram or Holter parameters, but there was a suggestion of more bradycardic events in the treated groups. Hypotension was less frequent in the treated patients compared with controls. CONCLUSIONS: In comparison with Ringer's acetate, Hemospan mildly elevates hepatic enzymes and lipase and is associated with less hypotension and more bradycardic events. The absence of a high frequency of serious adverse events suggests that further clinical trials should be undertaken.

Effect of ruboxistaurin on visual loss in patients with diabetic retinopathy.

OBJECTIVE: To evaluate the effect of ruboxistaurin, an orally administered protein kinase C beta (PKC beta) isozyme-selective inhibitor, on vision loss in patients with diabetes. DESIGN: Thirty-six-month, randomized, double-masked, placebo-controlled, parallel, multicenter trial. PARTICIPANTS: Six hundred eighty-five patients randomized at 70 clinical sites. METHODS: Ophthalmologic examination was performed at screening and at each 3-month visit. Retinopathy status was assessed every 6 months with Early Treatment Diabetic Retinopathy Study (ETDRS) standard 7-field 30 degrees color stereoscopic fundus photography. Levels of diabetic retinopathy and diabetic macular edema were determined by 2 independent graders masked to site and treatment assignment, with additional independent adjudication as required. Eligible patients had a best-corrected visual acuity (VA) score of > or =45 letters, retinopathy level > or = 47A and < or = 53E, and no prior panretinal photocoagulation in at least one eye. MAIN OUTCOME MEASURE: Effect of oral ruboxistaurin (32 mg/day) on reduction of sustained moderate visual loss (> or =15-letter decrease in ETDRS VA score maintained > or = 6 months) in patients with moderately severe to very severe nonproliferative diabetic retinopathy. RESULTS: Sustained moderate visual loss occurred in 9.1% of placebo-treated patients versus 5.5% of ruboxistaurin-treated patients (40% risk reduction, P = 0.034). Mean VA was better in the ruboxistaurin-treated patients after 12 months. Baseline-to-end point visual improvement of > or =15 letters was more frequent (4.9% vs. 2.4%) and > or =15-letter worsening was less frequent (6.7% vs. 9.9%) in ruboxistaurin-treated patients relative to placebo (P = 0.005). When clinically significant macular edema was >100 microm from the center of the macula at baseline, ruboxistaurin treatment was associated with less frequent progression of edema to within 100 microm (68% vs. 50%, P = 0.003). Initial laser treatment for macular edema was 26% less frequent in eyes of ruboxistaurin-treated patients (P = 0.008). CONCLUSION: Oral ruboxistaurin treatment reduced vision loss, need for laser treatment, and macular edema progression, while increasing occurrence of visual improvement in patients with nonproliferative retinopathy.

CJC-1131. ConjuChem.

ConjuChem is developing CJC-1131, a drug affinity complex conjugate of glucagon-like peptide 1 for the potential treatment of type 2 diabetes. In August 2003, a phase I/II trial was completed and a phase II trial was expected to begin in October.